The aim of the study reported here was to evaluate the performance and variability of transgenic potato clones. For the genetic transformation the Polish potato cultivar Irga was chosen in order to improve resistance to a necrotic strain of potato virus Y (PVYN ) by introducing to its genome a truncated gene coding PVYN replicase (in sense and antisense orientations). Transgenic plants and clones derived from them were propagated and the obtained tubers were planted in a replicated field trial. The several agronomic and morphological traits were evaluated and compared with measurements for non-transgenic control plants. The traits of transgenic clones showed a much greater variability than non-transgenic plants. The variability depended on the type of the introduced construct (in this case it was the orientation of the construct). None of the transgenic clones turned out to be completely true to type and resistant to PVYN, but some resistant clones expressed deviations in a small proportion of the traits. Usually, deviations were observed for those traits whose inheritance is characterised by a large environmental component. Genetic transformation is an effective method for introducing resistance. However, the method causes a great variability, which makes selection among transgenic clones a necessary step in breeding of an improved transgenic cultivar. Such selection has many similarities with selection done among traditionally obtained clones.
The Ns gene confers resistance of potato to Potato virus S (PVS). Sixteen German and Dutch potato cultivars, all registered in Poland, were found to be susceptible to PVS infection. However, scoring of the cultivars for the presence of the Ns-linked SCAR marker SC811454 revealed additional amplicons with a similar electrophoretic migration rate as that of SC811454, which resulted in ambiguous determination of the genotype at the Ns locus. MboI or FokI treatment of the PCR products allowed to detect their Ns-unspecificity in PVS-susceptible potato cultivars.
Preliminary investigations were carried out on the transformation of high susceptibility tobacco cultivars. The plants were transformed by being infected with Agrobacterium tumefaciens with binary PROK2- derived plasmids carrying a PVY casette in the sense and antisense orientations and with a plasmid carrying the lettuce mosaic virus (LMV) coat protein gene. Kanamycin resistant plants were obtained following transformation. PCR testing of a selected group of regenerants revealed the presence of a transgene. Disease symptoms were absent from 52% of transgenic plants inoculated with PVYN. This was confirmed by ELISA values.In the T1 generation of transgenic plants, 510 (78%) showed no symptoms after inoculation and 155 (22%) showed typical PVYN symptoms. Among 510 symptomless plants 213 (36%) were selected as highly resistant, 207 (31%) as partially resistant and 89 (11%) as tolerant. Forty kanamycin resistant plants of T2 generation were tested with PVYN. Two plants showed vein necrosis, five - vein clearing and 33 plants showed no disease symptoms. ELISA tests allowed to identify 33 plants (84,6%) totally resistant to PVYN. The preliminary data reported here showed more resistant plants in T2 generations.
Inter-simple sequence repeat (ISSR) polymorphism was used for finding markers linked to the Ns gene, responsible for a resistance of potato (Solanum tuberosum L.) to potato virus S (PVS). The ISSR markers UBC811660 and UBC811950 were found to be linked to Ns. Linkage distances were estimated to be 2.6 cM and 6.6 cM, respectively. UBC811660 showed high accuracy for detection of PVS resistance in diploid potato clones. In tetraploids, among seventeen studied genotypes containing the resistance gene, this marker was revealed in eleven. UBC811660 can be a powerful tool for detection of genotypes carrying the Ns gene in diploid potato breeding programmes.
In recent years a new subgroup of necrotic potato virus Y (PVY) isolates has spread in the Polish fields. To counter this infection, the PVY resistance was introduced into selected potato and tobacco cultivars. Plants were transformed by agroinfection with pROK2 derived binary plasmids, carrying an appropriate fragment of the PVY genome (patent applied for). Kanamycin resistant plants were screened by PCR for the presence of PVY cDNA inserts, positive transformants were tested for virus resistance by inoculation with sap from infected plants, followed by observations of disease symptoms and virus accumulation analysis. Several resistant potato and tobacco clones were identified and their phenotypes were preliminarily characterised.
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